Methods for electronic fluorescent perturbation for analysis and electronic perturbation catalysis for synthesis

ABSTRACT

Methods for catalyzing cleavage of a bond with an electric field device. In one method, a catalytic peptide having a first reactive group and a second reactive group is coupled to an electrode. The catalytic peptide is then contacted with a solution containing a substrate. The first reactive group reacts with the substrate to form a first intermediate. The second reactive group then reacts with the first intermediate to form a positively-charged second intermediate having an acyl bond and a negatively-charged first reactive group. An electronic pulsing sequence is then applied to the electrode to separate the negatively-charged first reactive group and the positively-charged second intermediate. The second intermediate is then reacted by acyl transfer to cleave the acyl bond. The first reactive group may be a sulfhydryl or deprotonated sulfhydryl. The second reactive group may be an imidazole. The substrate may contain an ester or amide bond.

RELATED APPLICATION INFORMATION

This application is a continuation of application Ser. No. 10/623,080,filed Jul. 18, 2003, which is a continuation application of applicationSer. No. 09/496,864, filed Feb. 2, 2000, now abandoned, which is acontinuation application of application Ser. No. 08/855,058, filed May14, 1997, entitled “Methods for Electronic Fluorescent Perturbation forAnalysis and electronic Perturbation Catalysis for Synthesis,” issued asU.S. Pat. No. 6,048,690, all of which are incorporated herein byreference as if fully set forth herein.

FIELD OF THE INVENTION

This invention relates to systems, devices, methods, and mechanisms forperforming multi-step molecular biological analysis, nucleic acidhybridization reactions, nucleic acid sequencing, and the catalysis ofbiomolecular, organic and inorganic reactions. More particularly, themolecular biological type analysis involves electronic fluorescentperturbation mechanisms for the detection of DNA hybrids, pointmutations, deletions or repeating sequences in nucleic acidhybridization reactions, electronic fluorescent perturbation mechanismsfor sequencing of DNA and RNA molecules, and electric field basedcatalytic mechanisms for biomolecular, biopolymer and other chemicalreactions.

BACKGROUND OF THE INVENTION

Molecular biology comprises a wide variety of techniques for theanalysis of nucleic acid and protein. Many of these techniques andprocedures form the basis of clinical diagnostic assays and tests. Thesetechniques include nucleic acid hybridization analysis, restrictionenzyme analysis, genetic sequence analysis, and the separation andpurification of nucleic acids and proteins (See, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989).

Most of these techniques involve carrying out numerous operations (e.g.,pipetting, centrifugations, electrophoresis) on a large number ofsamples. They are often complex and time consuming, and generallyrequire a high degree of accuracy. Many a technique is limited in itsapplication by a lack of sensitivity, specificity, or reproducibility.For example, these problems have limited many diagnostic applications ofnucleic acid hybridization analysis.

The complete process for carrying out a DNA hybridization analysis for agenetic or infectious disease is very involved. Broadly speaking, thecomplete process may be divided into a number of steps and substeps. Inthe case of genetic disease diagnosis, the first step involves obtainingthe sample (blood or tissue). Depending on the type of sample, variouspre-treatments would be carried out. The second step involves disruptingor lysing the cells, which then release the crude DNA material alongwith other cellular constituents. Generally, several sub-steps arenecessary to remove cell debris and to purify further the crude DNA. Atthis point several options exist for further processing and analysis.One option involves denaturing the purified sample DNA and carrying outa direct hybridization analysis in one of many formats (dot blot,microbead, microliter plate, etc.). A second option, called Southernblot hybridization, involves cleaving the DNA with restriction enzymes,separating the DNA fragments on an electrophoretic gel, blotting to amembrane filter, and then hybridizing the blot with specific DNA probesequences. This procedure effectively reduces the complexity of thegenomic DNA sample, and thereby helps to improve the hybridizationspecificity and sensitivity. Unfortunately, this procedure is long andarduous. A third option is to carry out the polymerase chain reaction(PCR) or other amplification procedure. The PCR procedure amplifies(increases) the number of target DNA sequences. Amplification of targetDNA helps to overcome problems related to complexity and sensitivity ingenomic DNA analysis. All these procedures are time consuming,relatively complicated, and add significantly to the cost of adiagnostic test. After these sample preparation and DNA processingsteps, the actual hybridization reaction is performed. Finally,detection and data analysis convert the hybridization event into ananalytical result.

The steps of sample preparation and processing have typically beenperformed separate and apart from the other main steps of hybridizationand detection and analysis. Indeed, the various substeps comprisingsample preparation and DNA processing have often been performed as adiscrete operation separate and apart from the other substeps.Considering these substeps in more detail, samples have been obtainedthrough any number of means, such as obtaining of full blood, tissue, orother biological fluid samples. In the case of blood, the sample isprocessed to remove red blood cells and retain the desired nucleated(white) cells. This process is usually carried out by density gradientcentrifugation. Cell disruption or lysis is then carried out, preferablyby the technique of sonication, freeze/thawing, or by addition of lysingreagents. Crude DNA is then separated from the cellular debris by acentrifugation step. Prior to hybridization, double-stranded DNA isdenatured into single-stranded form. Denaturation of the double-strandedDNA has generally been performed by the techniques involving heating(>Tm), changing salt concentration, addition of base (NaOH), ordenaturing reagents (urea, formamide, etc.). Workers have suggesteddenaturing DNA into its single-stranded form in an electrochemical cell.The theory is stated to be that there is electron transfer to the DNA atthe interface of an electrode, which effectively weakens thedouble-stranded structure and results in separation of the strands. See,generally, Stanley, “DNA Denaturation by an Electric Potential”, U.K.patent application 2,247,889 published Mar. 18, 1992.

Nucleic acid hybridization analysis generally involves the detection ofa very small number of specific target nucleic acids (DNA or RNA) withan excess of probe DNA, among a relatively large amount of complexnon-target nucleic acids. The substeps of DNA complexity reduction insample preparation have been utilized to help detect low copy numbers(i.e. 10,000 to 100,000) of nucleic acid targets. DNA complexity isovercome to some degree by amplification of target nucleic acidsequences using polymerase chain reaction (PCR). (See, M. A. Innis etal, PCR Protocols: A Guide to Methods and Applications, Academic Press,1990). While amplification results in an enormous number of targetnucleic acid sequences that improves the subsequent direct probehybridization step, amplification involves lengthy and cumbersomeprocedures that typically must be performed on a stand alone basisrelative to the other substeps. Substantially complicated and relativelylarge equipment is required to perform the amplification step.

The actual hybridization reaction represents the most important andcentral step in the whole process. The hybridization step involvesplacing the prepared DNA sample in contact with a specific reporterprobe, at a set of optimal conditions for hybridization to occur to thetarget DNA sequence. Hybridization may be performed in any one of anumber of formats. For example, multiple sample nucleic acidhybridization analysis has been conducted on a variety of filter andsolid support formats (See G. A. Beltz et al., in Methods in Enzymology,Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press,New York, Chapter 19, pp. 266-308, 1985). One format, the so-called “dotblot” hybridization, involves the non-covalent attachment of target DNAsto filter, which are subsequently hybridized with a radioisotope labeledprobe(s). “Dot blot” hybridization gained wide-spread use, and manyversions were developed (see M. L. M. Anderson and B. D. Young, inNucleic Acid Hybridization—A Practical Approach, B. D. Hames and S. J.Higgins, Eds., IRL Press, Washington, D.C. Chapter 4, pp. 73-111, 1985).It has been developed for multiple analysis of genomic mutations (D.Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for thedetection of overlapping clones and the construction of genomic maps (G.A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).

New techniques are being developed for carrying out multiple samplenucleic acid hybridization analysis on micro-formatted multiplex ormatrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp.1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). Thesemethods usually attach specific DNA sequences to very small specificareas of a solid support, such as micro-wells of a DNA chip. Thesehybridization formats are micro-scale versions of the conventional “dotblot” and “sandwich” hybridization systems.

The micro-formatted hybridization can be used to carry out “sequencingby hybridization” (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991;W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of allpossible n-nucleotide oligomers (n-mers) to identify n-mers in anunknown DNA sample, which are subsequently aligned by algorithm analysisto produce the DNA sequence (R. Drmanac and R. Crkvenjakov, YugoslavPatent Application No. 570/87, 1987; R. Drmanac et al., 4 Genomics, 114,1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1992; andR. Dramanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13,1993).

There are two formats for carrying out SBH. The first format involvescreating an array of all possible n-mers on a support, which is thenhybridized with the target sequence. The second format involvesattaching the target sequence to a support, which is sequentially probedwith all possible n-mers. Both formats have the fundamental problems ofdirect probe hybridizations and additional difficulties related tomultiplex hybridizations.

Southern, United Kingdom Patent Application GB 8810400, 1988; E. M.Southern et al., 13 Genomics 1008, 1992, proposed using the first formatto analyze or sequence DNA. Southern identified a known single pointmutation using PCR amplified genomic DNA. Southern also described amethod for synthesizing an array of oligonucleotides on a solid supportfor SBH. However, Southern did not address how to achieve optimalstringency condition for each oligonucleotide on an array.

Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used thesecond format to sequence several short (116 bp) DNA sequences. TargetDNAs were attached to membrane supports (“dot blot” format). Each filterwas sequentially hybridized with 272 labeled 10-mer and 11-meroligonucleotides. A wide range of stringency condition was used toachieve specific hybridization for each n-mer probe; washing timesvaried from 5 minutes to overnight, and temperatures from 0° C. to 16°C. Most probes required 3 hours of washing at 16° C. The filters had tobe exposed for 2 to 18 hours in order to detect hybridization signals.The overall false positive hybridization rate was 5% in spite of thesimple target sequences, the reduced set of oligomer probes, and the useof the most stringent conditions available.

A variety of methods exist for detection and analysis of thehybridization events. Depending on the reporter group (fluorophore,enzyme, radioisotope, etc.) used to label the DNA probe, detection andanalysis are carried out fluorometrically, calorimetrically, or byautoradiography. By observing and measuring emitted radiation, such asfluorescent radiation or particle emission, information may be obtainedabout the hybridization events. Even when detection methods have veryhigh intrinsic sensitivity, detection of hybridization events isdifficult because of the background presence of non-specifically boundmaterials.

In the many applications of DNA hybridization for research anddiagnostics, the most difficult analysis involve the differentiation ofa single base mismatch from a match target sequence. This is because theanalysis involves discriminating a small difference in one hybridizedpair, the mismatch, from the match. The teachings of this invention areof particular relevance to these problems.

SUMMARY OF THE INVENTION

As a main aspect of this invention, it has been surprisingly discoveredthat the fluorescence signal obtained during the electronic denaturationor dehybridization of DNA hybrids is perturbed at or around theelectronic power (current and voltage) levels which are associated withthe denaturation or dehybridization process. In one embodiment, thefluorescence signal perturbation phenomena appears as a rise or spike influorescence intensity prior to dehybridization of a fluorescent labeledprobe from a capture sequence attached to the microlocation test site.The power level, amplitude and slope of this fluorescence spike provideanalytical tools for diagnosis. The combination of the fluorescenceperturbation with other measurements also indicative of thehybridization match/mismatch state, such as consideration of theelectronic melting (50% fluorescence decrease during electronicstringency control) can in combination provide a more efficient andreliable hybridization match/mismatch analysis.

In general, this controlled dehybridization or electronic stringencyprocess results in a significant differential between the finalfluorescent intensity values for the match versus the mismatch sequence.This difference in fluorescent intensity values is used to determine adiscrimination ratio, which confirms and identifies that a particularmismatch was present in the sample.

It has been discovered that the fluorescent perturbation effect (FPE)provides a powerful analytical tool for DNA hybridization analysis,particularly for the near instantaneous, e.g., less than one minute, andespecially less than 5 seconds, discrimination of match/mismatched DNAhybrids. Novel DNA sequencing applications are possible. New fluorescentdonor/acceptor/quencher energy transfer mechanisms are created. Newelectronic catalytic mechanisms are created.

In one aspect, this invention relates to using precisely controlledelectric or electrophoretic fields to cause or influence fluorophore orchromophore groups in special arrangements with molecular structures(such as nucleic acids), to produce rapid signal variations(perturbations) which correlate with and identify small differences inthese molecular structures. In a preferred method for hybridizationanalysis of a sample, an electronic stringency control device is used toperform the steps of: providing the sample, a first probe with afluorescent label and a second probe with a label under hybridizationconditions on the electronic stringency control device, forming ahybridization product, subjecting the hybridization product to anelectric field force, monitoring the fluorescence from the hybridizationproduct, and analyzing the fluorescent signal. The label preferablyserves as a quencher for the fluorescent label.

Most broadly, this invention relates to integrated microelectronicsystems, devices, components, electronic based procedures, electronicbased methods, electronic based mechanisms, and flurophore/chromophorearrangements for: (1) molecular biological and clinical diagnosticanalyses; (2) nucleic acid sequencing applications; and (3) for carryingout catalysis of biomolecular, organic, and inorganic reactions.

More specifically, the molecular biological and clinical diagnosticanalyses relate to the utilization of the electronic fluorescentperturbation based mechanisms for the detection and identification ofnucleic acid hybrids, single base mismatches, point mutations, singlenucleotide polymorphisms (SNPs), base deletions, base insertions,crossover/splicing points (translocations), intron/exon junctions,restriction fragment length polymorphisms (RFLPs), short tandem repeats(STRs) and other repeating or polymorphic sequences in nucleic acids.

More specifically, the nucleic acid sequencing applications involveutilization of the electronic fluorescent perturbation based mechanismsto elucidate base sequence information in DNA, RNA and in nucleic acidderivatives. Most particularly, to elucidate sequence information fromthe terminal ends of the nucleic acid molecules. This method achieveselectronic fluorescence perturbation on an electronic stringency controldevice comprising the steps of: locating a first polynucleotide and asecond polynucleotide adjacent the electronic stringency control device,the first polynucleotide and second polynucleotide being complementaryover at least a portion of their lengths and forming a hybridizationproduct, the hybridization product having an associated environmentalsensitive emission label, subjecting the hybridization product and labelto a varying electrophoretic force, monitoring the emission from thelabel, and analyzing the monitored emission to determine the electronicfluorescence perturbation effect.

More specifically, the catalytic reactions relate to the utilization ofelectronic based catalytic mechanisms for carrying out biomolecular,biopolymer, organic polymer, inorganic polymer, organic, inorganic, andother types of chemical reactions. Additionally, the electronic basedcatalytic mechanisms can be utilized for carrying out nanofabrication,and other self-assembly or self-organizational processes. This methodprovides for electronic perturbation catalysis of substrate molecules onan electronic control device containing at least one microlocationcomprising the steps of: immobilizing on the microlocation anarrangement of one or more reactive groups, exposing the reactive groupsto a solution containing the substrate molecules of interest, andapplying an electronic pulsing sequence which causes charge separationbetween the reactive groups to produce a catalytic reaction on thesubstrate molecules.

More generally, the present invention relates to the design,fabrication, and uses of self-addressable self-assemblingmicroelectronic integrated systems, devices, and components whichutilize the electronic mechanisms for carrying out the controlledmulti-step processing and multiplex reactions in a microscopic,semi-microscopic and macroscopic formats. These reactions include, butare not limited to, most molecular biological procedures, such as: (1)multiplex nucleic acid hybridization analysis in reverse dot blotformats, sandwich formats, homogeneous/heterogeneous formats,target/probe formats, in-situ formats, and flow cytometry formats; (2)nucleic acid, DNA, and RNA sequencing; (3) molecular biologicalrestriction reactions, ligation reactions, and amplification typereactions; (4) immunodiagnostic and antibody/antigen reactions; (5) celltyping and separation procedures; and (6) enzymatic and clinicalchemistry type reactions and assays.

In addition, the integrated systems, devices, and components whichutilize electronic based catalytic mechanisms are able to carry outbiomolecular, biopolymer and other types of chemical reactions: (1)based on electric field catalysis; and/or (2) based on multi-stepcombinatorial biopolymer synthesis, including, but not limited to, thesynthesis of polynucleotides and oligonucleotides, peptides, organicmolecules, bio-polymers, organic polymers, mixed biopolymers/organicpolymers, two and three dimensional nanostructures, and nanostructuresand micron-scale structures on or within silicon or other substratematerials.

Additionally, with respect to electronic fluorescent perturbationmechanisms, the present invention relates to unique intermolecular andintramolecular constructs and arrangements of chromophores,fluorophores, luminescent molecules or moities, metal chelates(complexes), enzymes, peptides, and amino acids, associated with nucleicacid sequences, polypeptide sequences, and/or other polymeric materials.Of particular importance being those constructs and arrangements offluorophores and chromophores which produce fluorescent energy transfer,charge transfer or mechanical mechanisms which can be modulated oraffected by electric or electrophoretic fields to produce fluorescent orluminescent signals which provide information about molecular structure.

With respect to the electronic catalytic mechanisms in homogeneous(solution) or heterogeneous (solution/solid support) formats, thepresent invention relates to unique intermolecular and intramolecularconstructs and arrangements of chromophores, fluorophores, luminescentmolecules or moities, metal chelates (complexes), enzymes, peptides, andamino acids, nucleophilic molecules or moities, electrophilic moleculesor moities, general acid or base catalytic molecules or moieties, andsubstrate binding site molecules and moities, associated with nucleicacid sequences, polypeptide sequences, other biopolymers, organicpolymers, inorganic polymers, and other polymeric materials.

Additionally, this invention relates to the utilization of electric orelectrophoretic fields to induce fluorescent perturbation basedmechanisms in arrangements of fluorophores and chromophores in solidstate or sol-gel state optoelectronic devices and optical memorymaterials.

It is therefore an object of this invention to provide for methods andsystems for improved detection and analysis of biological materials.

It is yet a further object of this invention to provide for methodswhich provide for the rapid and accurate discrimination between matchesand mismatches in nucleic acid hybrids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a plot of the relative fluorescent intensity as a function ofapplied power (microwatts) for a 20-mer oligomer duplex (100% AP).

FIG. 1B is a plot of the relative fluorescent intensity versus appliedpower (microwatts) for a 19-mer oligomer duplex (53% GC).

FIG. 2A is a graph of the relative fluorescent intensity verus appliedpower (microwatts) for a 20-mer oligomer duplex (100% AT).

FIG. 2B is a plot of the relative fluorescent intensity verus appliedpower (microwatts) for a 19-mer oligomer duplex (53% GC).

FIG. 3A shows a cross-sectional view of a mismatched test site having acapture probe, target DNA and a reporter probe.

FIG. 3B is a cross-sectional view of target DNA and a reporter probewith an associated fluorophore.

FIG. 3C is a graph of the fluorescent response graphing the relativefluorescent intensity as a function of time for a pulses sequence.

FIG. 4A is a cross-sectional view of a matched test site having acapture probe, target DNA and a reporter probe with an intercalatedfluorophore.

FIG. 4B is a cross-sectional view of target DNA and a reporter probewith an intercalating fluorophore.

FIG. 4C is a graph of the fluorescent response showing the relativefluorescence intensity as a function of time for a pulsed sequence.

FIG. 5 shows the fluorescent intensity (% remaining Fluorescein)profiles as a function of time (seconds) for a one base mismatch and amatch sequence for Ras G 22 mers during the basic electronicdehybridization process.

FIG. 6 shows the fluorescent intensity (% remaining fluorescence) as afunction of time (seconds) observed during the general electronicdehybridization of match/mismatch hybrids for the Ras and RCA5 (HLA)systems.

FIG. 7A shows a graph of the normalized fluorescent intensity versustime (seconds) for match/mismatch profiles exhibiting the oscillatingfluorescent perturbation effect.

FIG. 7B shows an expanded view graph of the first 12 seconds of thegraph of FIG. 7A.

FIG. 8A shows a schematic representation for the hybridized arrangementof the target probe and the Bodipy Texas Red labeled reporter probe, andthe position of the one base mismatch.

FIG. 8B shows a schematic representation of FIG. 8A, but where amismatch between the target and probe is present.

FIG. 9 shows a graph of the normalized fluorescent intensity as afunction of time (seconds) match/mismatch profiles exhibiting theoscillating fluorescent perturbation effect, in the presence of a secondprobe containing a quencher group (Malachite Green).

FIG. 10A shows a schematic representation for the hybridized arrangementof the target probe, the Bodipy Texas Red labeled reporter probe, andthe Malachite Green quencher probe.

FIG. 10B shows the schematic representation of FIG. 10A with a mismatchbetween the target and the probe.

FIG. 11A shows a schematic representation for the hybridized arrangementof a target probe, a labeled reporter probe and a quencher probe.

FIG. 11B shows the schematic representation of FIG. 11A with a mismatchbetween the target and probe.

FIG. 12 shows a sequence of steps for electronic perturbation catalysis.

DETAILED DESCRIPTION OF THE INVENTION

The APEX device as described in the various parent applications has beenutilized in novel ways resulting in methods which improve the analyticalor diagnostic capabilities of the device. It has been surprisinglydiscovered that the fluorescent signal is perturbed during theelectronic dehybridization of DNA hybrids. This method has particularapplication to DNA hybridization and single-base mismatch analysis.Specifically, during electronic dehybridization, also known asstringency control or electronic stringency control, a rise or spike inthe fluorescence intensity has been observed just prior to thedehybridization of the fluorescent labeled probes from capture sequencesattached to the APEX chip pad.

FIGS. 1A and 1B show the results of electronic denaturizationexperiments run on an APEX chip having 25 test microlocations with 80micron diameter utilizing platinum electrodes. For this use, the chipwas overlaid with a 1 micron thick avidin/agarose permeation layer. Two5′-labeled bodipy Texas Red (Ex 590 nm, EM 630 nm) target probes wereused in the experiments. The probe of FIG. 1A was a 20 mer(5′-BYTRAAATTTTAATATATAAT-3′) containing 100% AT, with a meltingtemperature (Tm) of 33° C. The probe of FIG. 1B was a 19 mer(5′BYTRCCACGTAGAACTGCTCATC-3′) containing 53% GC, with a meltingtemperature (Tm) of 54° C. (Melting temperature or Tm refers to thetemperature at which the dehybridization process is 50% complete). Theappropriate complementary biotinylated capture sequences were attachedto the avidin/agarose permeation layer over several of the test pads (onthe same chip). The capture probe density was ˜10⁸ probes per pad. Thefluorescent labeled target probes, at a concentration of ˜1.0 μM in 50mM sodium phosphate (pH 7.0), 500 mM NaCl were first hybridized to theattachment probes on the 5580 chips. The chips were then thoroughlywashed with 20 mM NaPO4 (pH 7.0).

Electronic denaturation was then carried out by biasing the test padnegative, and increasing the power to the test pad from ˜10⁻¹ microwatts(μW) to ˜2×10² microwatts (μW) over a 90 second time period. Three padswere tested for each of the target probes. The relative change influorescent intensity was plotted as a function of the increasing power.In general, the electrophoretic field, force or power necessary todehybridize a probe from its complementary sequence correlates with thebinding energy or Tm (melting temperature) for the DNA duplex. In aboveexperiments the overall power level (μW) necessary to dehybridize the19-mer probe with 53% GC probe (Tm of 54° C.) was higher than for the20-mer probe with 100% AT (Tm of 33° C.), that is, the equivalentelectronic melting point (Em) at which dehybridization is 50% completeis higher for the 53% GC probe. Also, the fluorescent perturbation(FIGS. 1A and 1B, circled region) for the 10-mer probe with 53% GC isobserved to be significantly different from that associated with the100% AT probe.

FIGS. 2A and 2B show the results of denaturation experiments run on theAPEX chip having 25 test microlocations with 20 micron deep wells to theunderlying platinum electrodes. The well structures on the chip werefilled with avidin/agarose composite, forming a 20 micron deeppermeation layer. The same fluorescent target probes, capture probes andprotocols were used in the deep well experiments as in the operation ofthe device resulting in the information of FIGS. 1A and 1B. As in thefirst experiments, the overall power (μW) necessary to dehybridize the19-mer probe with 53% GC (Tm of 54° C.), is higher than for the 20-merprobe with 100% AT (Tm of 33° C.). Also, the slope for the 100% AT probeis much shallower, then for the 53% GC probe. The fluorescentperturbation/spike phenomena is very pronounced for the 19-mer probewith 53% GC in the deep well experiments.

The fluorescent perturbation phenomena correlates well with the sequencespecificity of the dehybridization process. The power level (μW) value,amplitude and slope of the fluorescent spike are useful for many aspectsof hybridization analysis including single base mismatch analysis. Thefluorescent perturbation (Fp) value, namely those values associated withthe fluorescence perturbation, e.g., onset value, peak height and slope,combined with the electronic melting (Em) values, namely, thehalf-height value of fluorescence, provide significantly higherreliability and additional certainty to hybridization match/mismatchanalysis. By combining two or more analytical measurements, a moreeffective and precise determination may be made.

In the above experiments, the target probes were labeled with a BodipyTexas Red fluorophore in their 5′ terminal positions. While Bodipy TR isnot a particularly environmentally sensitive fluorophore it neverthelessshowed pronounced effects during electronic denaturation. Moreenvironmentally sensitive fluorophores may be used to obtain largerperturbations in their fluorescent properties during electronicdehybridization.

The placement of a sensitive fluorescent label in optimal proximity tothe initial denaturation site is preferred. By associating certainfluorescent labels in proximity to the denaturation site, as opposed tolabeling at the end of the target or probe, increased specificity andenhanced effects may result. As shown in FIGS. 3A and 4A, anintercalating fluorophore 10 may be disposed between a reporter probe 2and target DNA 4. FIG. 3A shows the condition in which the reporterprobe 2 is mismatched from the target DNA 4 by a mismatched base 6. Ineach of FIGS. 3A and 4A, the capture probe 8 serves to capture thetarget DNA 4, with the pad 12 providing the electrophoretic action.Preferably, the intercalating fluorophore 10 would be placed next to thesingle base mismatch site 6 (FIG. 3A). The intercalating typefluorescent label could be, for example, ethidium bromide and acridinederivatives, or any other known fluorescent labels consistent with theobjects of this device and its use.

FIGS. 3B and 4B show the condition of the reporter probe 2, the targetDNA 4 and the mismatch base site 6 after the application of a pulse atthe fluorescent perturbation value via the pad 12. The change fromintercalated to the non-intercalated environment would produce a majorchange in fluorescent signal intensity for certain labels like ethidium.

Furthermore, the use of a mismatch site directed fluorophore label doesnot require that the hybrid be completely denatured during the process.As shown in FIG. 3C and FIG. 4C, an analysis procedure is preferred inwhich an appropriate pulsed “Fp” power level is applied which causes amismatched hybridization site to partially denature and renaturerelative to a matched hybridization site. The procedure results in anoscillating fluorescent signal being observed for mismatch hybrid site,while the fluorescent signal for the matched hybrid site remainsunchanged. FIGS. 3C and 4C shows the relative fluorescent intensity as afunction of varied applied power. This procedure provides a highlyspecific and discriminating method for single base mismatch analysis.Additional advantages include: (1) longer probes (>20-mer) than thoseused in conventional hybridization procedures can be used in thisprocess, (2) Probe specificity is more determined by placement of thefluorescent label (particularly for single base mismatches), and (3) asthe procedure does not require complete denaturation of the hybridstructures, each sample can be analyzed repetitively for providing ahigher statistical significant data, such as through standard averagingtechniques.

Referring to FIG. 5, in the process of carrying out electronic DNAhybridization and selective dehybridization (by electronic stringency)on active DNA chip devices (e.g., on an APEX chip), it was surprisinglydiscovered that the fluorescence signal from labeled reporter probes ortarget DNAs was perturbed during the initiation of electronicdehybridization at or around the electronic power levels (current andvoltage) associated with that dehybridization process. Specifically,this fluorescence signal perturbation shows itself often as a rise orspike in the fluorescence intensity prior to dehybridization of thefluorescent labeled probe sequence from the DNA sequence attached to themicroscopic test site (microlocation) on the DNA chip surface. The mainregion of fluorescence perturbation is shown in the dashed circle.

The fluorescent perturbation effect (FPE) is usually most pronounced fora one base mismatched probe sequence relative to the match probesequence. In the general electronic hybridization and dehybridizationprocedure, the precisely controlled electronic stringency processresults in a significant differential between the final fluorescentintensity values for the match versus the mismatch sequence. Themismatch sequence is more effectively dehybridized and more rapidlyremoved from the test location than the match sequence. In the generalelectronic hybridization and dehybridization process this difference influorescent intensity values is used to determine a discriminationratio, which confirms and identifies that a particular mismatch waspresent in the sample. The particular parameters of electric fieldstrength (current/voltage), solution conductivity, electrode geometryand pulsing time used to produce this selective dehybridization betweenthe match and the mismatch occur at what is called the electronicmelting temperature (Etm). The electronic dehybridization and stringencyprocess allows match/mismatch discriminations to be carried out veryrapidly (within substantially 20 to 60 seconds), compared with theclassical hybridization stringency process, which involves temperaturecontrol and stringent washing procedures, which can take hours tocomplete. The single base pair mismatch (single BPM) sequence isobserved to decrease faster than the match sequence allowing one toobtain a match/mismatch discrimination ratio for the pair.

Initial observations of the fluorescent perturbation effect (FPE), whichoccurs almost immediately upon initiation of the electronicdehybridization process, indicated that it was possible to use the FPEas a way to distinguish match/mismatched DNA hybrids even more rapidly,typically in less than a minute, and most preferably in several secondsor less. Another very powerful and novel feature of the FPE is that thistechnique does not require the removal of the probe or target sequencein order to discriminate a match from the mismatch hybrid, whereas thegeneral electronic dehybridization process and classical hybridizationtechniques typically require the removal of the mismatch sequencerelative to the matched sequence. A further advantage of the FPEtechnique is that probes of any size can potentially be used formatch/mismatch hybrid discriminations or other applications. Longerprobes sequences can provide overall better hybridization stability andselectivity.

Further investigations of the fluorescent perturbation effect hasrevealed other aspects and advantages of this unique phenomena whichinclude: (1) that the amplitude, frequency, and slope of thisfluorescent signal can provide a powerful analytical tool for othertypes of DNA hybridization analysis, in addition to the nearinstantaneous discrimination of single base mismatched DNA; (2) thatmultiple probe systems, involving a quencher probe and fluorescentacceptor probe (and donor probes), can be used to further enhance theFPE technique; (3) that a variety of electronic pulsing sequences (DCand AC variations) can be developed which further improve and broadenthe scope of FPE based analysis of DNA and other molecular structures;(4) that the electronic fluorescence perturbation mechanism could leadto DNA sequencing applications; (5) that new arrangements of fluorescentdonor/acceptor/quencher groups could be created for improved energytransfer mechanisms and applications; and (6) that novel electroniccatalytic mechanisms could be created. These are the subjects of thisinvention.

The basic fluorescent perturbation effect occurs generally upon theinitiation of electronic denaturation of match and mismatch hybridpairs. In the case of the Ras (ras oncogene) hybrids in FIG. 5, themismatch nucleotide is located approximately in the middle of the probesequence, and the fluorescent label (Bodipy Texas Red) is covalentlyattached to the terminal position of the oligonucleotide sequence,approximately 10 bases from the mismatched nucleotide (see Example 1,below). Upon initiation of dehybridization process the fluorophoreresponds to the changing environment of the dehybridizing DNA strands bybrightening. Generally, most fluorophores are somewhat sensitive totheir local physical, chemical, and thermal environments; and a numberof fluorophores are found to be extremely sensitive to changes in theirenvironment. Environmental parameters such as hydrophilicity,hydrophobicity, pH, electrostatic charge, and Van der Waalsinteractions, can cause changes in the fluorescent intensity (quantumyield), the excitation/emission spectrum, and/or the fluorescent lifetime. Many of these environmental parameters are believed to change dueto some or all of: (1) the disruption of the double-stranded DNAstructure; (2) the effect of a DC or AC electric field and/or theelectrophoretic effects on the fluorophore itself; (3) the effect of aDC or AC electric field and/or the electrophoretic effects on thefluorophore/DNA structure, which has its own unique set of interactionsthat can depend upon base sequence (AT or GC rich areas), and whetherthe fluorophore is associated with a double or single-stranded form ofthe nucleic acid; and/or (3) changes in the local electrochemicalenvironment. It does appear that initial destabilization of thedouble-stranded structure is most important to the process. This isbecause the effect on the mismatch is more pronounced than for thematch, both of which are present in the same general environment.

It is believed that the subtle fluorophore/DNA structural interactionsare also very important. This is the basis for DNA sequencing techniquesdisclosed herein.

FIG. 6 shows some further examples of the fluorescent perturbationeffect observed during the general electronic dehybridization andstringency process for match/mismatch hybrids for the Ras and RCA5 (HLA)systems (see Example 2, below). The effect again is observed for boththe Ras and RCA5 mismatch sequences, being particularly pronounced forRCA5 hybrid pair.

In general electronic hybridization and stringency experiments, thereporter or target probes are typically labeled with a Bodipy Texas Redfluorophore in their 5′ (or 3′) terminal positions. While Bodipy TR isnot a particularly environmentally sensitive fluorophore it neverthelessshowed pronounced effects during electronic dehybridization process.More environmentally sensitive fluorophores may be used to obtain largerperturbations in their fluorescent properties during FPE process. By wayof example, these fluorophores and chromophores include: other Bodipydye derivatives, ethidiums (in particular derivatized forms of ethidiumdyes which can be covalently attached to DNA), or other intercalatingfluorophores (which are or can be derivatized for attachment to DNA,acridines, fluoresceins, rhodoamines, Texas Red (sulforhodamine 101),Cy3 and Cy5 dyes, Lucifer Yellow, and Europium and Terbium chelate dyederivatives, IR144 and far red laser dyes and derivatives. Otherfluorophores, chromophores and dyes consistent with the methods andobjects of these inventions may be utilized.

In general, any dye which is sensitive to the environmental parameterssuch as hydrophilicity, hydrophobicity, pH, electrostatic charge, Vander Waals interactions, etc., that can cause changes in the fluorescentintensity (quantum yield), the excitation/emission spectrum, and/or thefluorescent life time, are potentially useful for FPE applications. Moreparticularly useful, are those fluorophores, chromophores, or dyes whichhave properties which change or are perturbed due to the following.

(1) The initial disruption or destabilization of the double-stranded DNAstructure. This is optionally just near the terminal position of the DNAstructure where the fluorophore is located.

(2) The effects of the DC or AC electric field (or electrophoreticfield) on the fluorophore itself. Of importance would be whether thefluorophore is neutral or charged, and whether the net charge ispositive or negative. The net charge could strongly influence theperturbation effect, particularly if the fluorophore were positivelycharged. In this case, the fluorophore would tend to move in an oppositedirection relative to the rest of the DNA molecule when an electricfield is applied.

(3) The effect of the DC or AC electric field (or electrophoretic field)on the fluorophore/DNA interaction itself. Again, whether thefluorophore was neutral, net positive, or net negatively charged wouldhave a pronounced effect on the nature and stability of thefluorophore/DNA interaction.

(4) The general spectral properties and robustness of the dye are alsoimportant. For example, the excitation and emission maxima, the Stokesshift, and the resistance to fading under excitation conditions are alsoimportant. Of particular usefulness would be those dyes which haveexcitation maxima at or above 480 nm, and emissions at or above 520 nm,and Stokes shifts of more than 20 nm. More useful, would be those dyeswhich have excitation maxima at or above 590 nm, and emissions at orabove 620 nm, and Stokes shifts of more than 20 nm. Most useful, wouldbe those dyes which have excitation maximum at or above 650 nm, andemissions at or above 670 nm, and Stokes shifts of more than 20 nm.

The placement of a sensitive fluorophore or chromophore label orreporter in optimal proximity to the initial destabilization or basemismatch site is important for achieving the electronic fluorescentperturbation effect (FPE). The preferred arrangements would be to havethe fluorophore or chromophore within 0 to 10 bases of the initialdestabilization or base mismatch site. The most preferred arrangementswould be to have the fluorophore or chromophore within 0 to 5 bases ofthe initial destabilization or base mismatch site.

It should be kept in mind, that when a fluorophore or chromophore groupis at the terminal position (5′ or 3′) of a DNA sequence which ishybridized to a complementary sequence, the group is already located insome sense at a “destabilized” site relative to the rest of thehybridized structure. This is because the terminal or end positions of ahybrid structure are less stable (the strands are opening and closing orfraying) relative to the internal hybridized sequence. One importantaspect of this invention is to design the probe sequences such that theynow position the further destabilizing base mismatch nucleotide site (inthe target or probe sequence), so that upon hybridization the basemismatch is in closer proximity to the terminal fluorophore orchromophore group or groups. By associating the destabilization site incloser proximity to the terminal fluorophore or chromophore group(s), itis possible to utilize electronic pulsing sequences which producefluorescent perturbation effects which correlate well with molecularstructure, i.e., detect and identify point mutations, base deletions,base insertion, nucleotide repeat units, and other features important toDNA analysis.

Additional advantages to the FPE technique include: (1) the ability toutilize longer probes (>20-mer) than those used in conventionalhybridization procedures, (2) that probe specificity can be determinedby placement of the fluorophore or chromophore label (particularly forsingle base mismatches), and (3) FPE technique does not requiredehybridization or removal of the mismatched probe sequence from thesystem; therefore, each sample can be analyzed repetitively providing ahigher statistical significant to data, such as through signal averagingtechniques.

Most particularly, this invention relates to using precisely controlledAC or DC electric fields or electrophoretic fields to affect orinfluence fluorophore or chromophore groups in special arrangements withmolecular structures (such as nucleic acids), to produce rapid signalvariations (perturbations) which correlate with and identify smalldifferences in these molecular structures.

Most broadly, this invention relates to integrated microelectronicsystems, devices, components, electronic based procedures, electronicbased methods, electronic base mechanisms, and fluorophore/chromophorearrangements for: (1) molecular biological and clinical diagnosticanalyses; (2) nucleic acid sequencing applications; and (3) for carryingout catalysis of biomolecular, organic, and inorganic reactions.

More specifically, the molecular biological and clinical diagnosticanalyses relate to the utilization of the electronic fluorescentperturbation based mechanisms for the detection and identification ofnucleic acid hybrids, single base mismatches, point mutations, singlenucleotide polymorphisms (SNPs), base deletions, base insertions,crossover/splicing points (translocations), intron/exon junctions,restriction fragment length polymorphisms (RFLPs), short tandem repeats(STRs) and other repeating or polymorphic sequences in nucleic acidacids.

More specifically, the nucleic acid sequencing applications involveutilization of the electronic fluorescent perturbation based mechanismsto elucidate base sequence information in DNA, RNA, and in nucleic acidderivatives. Most particularly, to elucidate sequence information fromthe terminal ends of the nucleic acid molecules.

More specifically, the catalytic reactions relate to the utilization ofelectronic based catalytic mechanisms for carrying out biomolecular,biopolymer, organic polymer, inorganic polymer, organic, inorganic, andother types of chemical reactions. Additionally, the electronic basedcatalytic mechanisms can be utilized for carrying out nanofabrication,and other self-assembly or self-organizational processes. Moregenerally, the present invention relates to the design, fabrication, anduses of self-addressable self-assembling microelectronic integratedsystems, devices, and components which utilize the electronic mechanismsfor carrying out the controlled multi-step processing and multiplexreactions in a microscopic, semi-microscopic and macroscopic formats.These reactions include, but are not limited to, most molecularbiological procedures, such as: (1) multiplex nucleic acid hybridizationanalysis in reverse dot blot formats, sandwich formats,homogeneous/heterogeneous formats, target/probe formats, and in-situformats, and flow cytometry formats; (2) nucleic acid, DNA, and RNAsequencing; (3) molecular biological restriction reactions, ligationreactions, and amplification type reactions; (4) immunodiagnostic andantibody/antigen reactions; (5) cell typing and separation procedures;and (6) enzymatic and clinical chemistry type reactions and assays.

In addition, the integrated systems, devices, and components whichutilize electronic based catalytic mechanisms are able to carry outbiomolecular, biopolymer and other types of chemical reactions: (1)based on electric field catalysis; and/or (2) based on multi-stepcombinatorial biopolymer synthesis, including, but not limited to, thesynthesis of polynucleotides and oligonucleotides, peptides, organicmolecules, biopolymers, organic polymers, mixed biopolymers/organicpolymers, two and three dimensional nanostructures, and nanostructuresand micron-scale structures on or within silicon or other substratematerials.

Additionally, with respect to electronic fluorescent perturbationmechanisms, the present invention relates to unique intermolecular andintramolecular constructs and arrangements of chromophores,fluorophores, luminescent molecules or moities, metal chelates(complexes), enzymes, peptides, and amino acids, associated with nucleicacid sequences, polypeptide sequences, and/or other polymeric materials.Of particular importance being those constructs and arrangements offluorphores and chromophores which produce fluorescent energy transfer,charge transfer or mechanical mechanisms which can be modulated oraffected by the AC or DC electric fields or electrophoretic fields toproduce fluorescent or luminescent signals which provide informationabout molecular structure.

With respect to the electronic catalytic mechanisms in homogeneous(solution) or heterogeneous (solution/solid support) formats, thepresent invention relates to unique intermolecular and intramolecularconstructs and arrangements of chromophores, fluorophores, luminescentmolecules or moities, metal chelates (complexes), enzymes, peptides, andamino acids, nucleophilic molecules or moities, electrophilic moleculesor moities, general acid or base calalytic molecules or moieties, andsubstrate binding site molecules and moities, associated with nucleicacid sequences, polypeptide sequences, other biopolymers, organicpolymers, inorganic polymers, and other polymeric materials.

Additionally, this invention relates to the utilization of electric orelectrophoretic fields to induce fluorescent perturbation basedmechanisms in arrangements of fluorophores and chromophores in solidstate or sol-gel state optoelectronic devices and optical memorymaterials.

FPE with a Single Fluorophore

FIG. 7A shows a graph of the normalized match/mismatch profilesexhibiting the oscillating fluorescent perturbation effect for a probewith a single fluorescent reporter group. A pronounced difference isobserved between the match and the mismatch hybrids. The match andmismatch hybrid pairs have the mismatched nucleotide located two basesfrom the Bodipy Texas Red fluorescent reporter group which is attachedto the 3′-terminal position of the reporter probe. The x-axis of thegraph is seconds, and the y-axis is relative fluorescent intensityunits. The electronic pulse sequence used was 500 nA for 0.5 secondson/0.75 second off, run for 30 seconds (see Example 3). In this examplethe match and mismatch hybrid pairs have the mismatched nucleotidelocated two bases from the Bodipy Texas Red fluorescent reporter groupwhich is attached to the 3′-terminal position of the reporter probe.

FIG. 7B now shows an expanded view graph of the first 12 seconds for thenormalized match/mismatch profiles exhibiting the oscillatingfluorescent perturbation effect. A very pronounced difference isobserved in the first few seconds after the pulse sequence is initiated,after which the match and the mismatch continue to oscillate atdifferent amplitudes. It is believed that the higher amplitudeoscillation by the match is due to the faster and more efficientrehybridization by the fully complementary (match) sequence relative toa non-fully complementary sequence (mismatch). This faster “snap-back”of the match relative to the mismatch may be used to distinguish thosecases. FIG. 7B shows that the upon initiation of the DC pulse sequencethat the fluorescent intensity for the mismatch rises rapidly, while thefluorescent intensity for the match actually decreases momentarily. Themismatch and the match then seem to come into phase, but oscillate atdifferent amplitudes. It is such pronounced differences which allow theFPE to be used to differentiate between the match and mismatched DNAstructures.

FIGS. 8A and 8B show a schematic representation for the hybridizedarrangement of the target probe and the Bodipy Texas Red labeledreporter probe, and the position of the one base mismatch (FIG. 8B). Themismatched nucleotide is located two bases from the Bodipy Texas Redfluorescent reporter group which is attached to the 3′-terminal positionof the reporter probe. The most preferred arrangements for carrying outFPE techniques with a single fluorophore would be to have it locatedwithin 0 to 5 bases of the mismatched location (see Example 3, below).

FPE with Multiple Fluorophore/Chromophore Arrangements

FIG. 9 shows a graph of the normalized match/mismatch profilesexhibiting the oscillating fluorescent perturbation effect, in thepresence of a second probe containing a quencher group (MalachiteGreen). A pronounced difference is observed between the match and themismatch hybrids upon application of the electric field. There isimmediately a very large increase in fluorescent intensity due to theloss of the quenching effect upon initiation of the electric field.After the “de-quenching” the match and the mismatch continue tooscillate at different amplitudes. This represent just one example ofhow a unique fluorophore/chromophore arrangement can be used to enhanceor improve the FPE technique. Additionally, this represents an exampleof how a unique energy transfer or quenching mechanism can be designed,which responds to a DC pulsing electric field (electrophoretic field),and produces a unique fluorescent response (a dramatic increase inintensity). It is also disclosed in this invention, that AC electricfields (including high frequencies>100 Hz), would have fluorescentperturbation effects which would be useful for analysis of molecularstructures, in particular for DNA hybridization analysis.

In the example shown in FIG. 9, the match and mismatch hybrid pairs havethe mismatched nucleotide located two bases from the Bodipy Texas Redfluorescent reporter group, which is attached to the 3′-terminalposition of the reporter probe. The second probe (quencher probe)hybridizes to the target sequence in such a way that it positions theMalachite Green quencher group (attached at the 5′-terminal position)within three bases of the Bodipy Texas Red fluorophore group on the3′-terminal position of the reporter probe. Upon hybridization, thequencher probe causes about a 40-50% decrease in the fluorescentintensity of the Bodipy Texas Red reporter (which is eliminated when theelectric field is applied). Other arrangements and quencher chromophorescould produce even better quenching and reduction of fluorescence fromthe reporter group. In FIG. 9, the x-axis of the graph is in seconds,and the y-axis is in relative fluorescent intensity units. Theelectronic pulse sequence used was 600 nA for 1.0 seconds on/1.5 secondoff, run for 30 seconds (see Example 4, below).

FIGS. 10A and 10B show a schematic representation for the hybridizedarrangement of the target probe, the Bodipy Texas Red labeled reporterprobe, and the Malachite Green quencher probe. The mismatched nucleotide(FIG. 10B) is located two bases from the fluorescent reporter group(Bodipy Texas Red) located on the terminal position of the reporterprobe. The second probe (quencher probe) hybridizes to the targetsequence in such a way that it positions the Malachite Green quenchergroup (attached at the 5′-terminal position) within three bases of theBodipy Texas Red fluorophore group on the 3′-terminal position of thereporter probe. Other useful fluorophore/chromophore forms andarrangements would include those in which the quencher probe is designedto be hybridized within 0 to 5 bases of the mismatch position.

Of particular usefulness for this invention is one of the preferredarrangement shown in FIGS. 11A and 11B. In this example, the first probe(a capture/quencher probe sequence) has two terminal functional groups,a 5′-terminal biotin group which allows the probe to be immobilized tothe surface (permeation layer) of a microlocation test site on an activeDNA chip or other hybridzation device. The second functional group beinga quencher group, (such as Malachite Green, Reactive Red, or otherquencher chromophore), which is at the 3′-terminal position of thecapture/quencher probe. The capture/quencher probes are madecomplementary to the match and mismatch point mutation sequences ofinterest. These probes allow the target DNA (RNA) sequence to becaptured by selective hybridization and immobilized on the microlocationtest site. The sequence is designed to optimally position the(potential) mismatched nucleotide within one to five bases of thequencher group. After the hybridization/capture of the target DNA (RNA)sequence, the second probe (acceptor reporter) is added and hybridizedto the immobilized target DNA/quencher probe. The acceptor/reporterprobe is labeled in its 5′-termininal position with a suitablefluorophore (Bodipy Texas Red, or other reporter group), and designed tohybridize to the target DNA sequence in such a away as to be optimallypositioned within 1 to 5 bases of the quencher group, where uponhybridization the acceptor reporter groups fluorescence is quenched.Upon application of the appropriate electronic DC pulsing sequence(current/on time/off time) an electric field is induced which causes thematch and mismatched hybrids to produce a fluorescent perturbationeffect and oscillations which allow them to be distinguished andidentified. It should be pointed out that the above hybridizationprocedure could also be carried out in a semi-homogeneous format, inwhich the target DNA sequence is first hybridized in solution with thereporter probe sequence, before hybridization to the immobilizedcapture/quencher probe. The above describes just some of the potentiallyuseful formats for PFE. It is important to realize that flexibility inchoosing various FPE techniques and formats will be advantageous forsuccessful broad area hybridization diagnostics. The scope of thisinvention also includes the utilization of the FPE processes describedabove, in highly multipexed formats on APEX DNA chips and array devices.

Additionally, the scope of this invention includes the use andincorporation of various donor/acceptor/quencher, mechanisms, probearrangements and hybridization formats which were described in ourphotonic patents (U.S. Pat. No. 5,532,129 and U.S. Pat. No. 5,565,322)and optical memory application (WO 95/34890). The novel electronicpulsing scenarios combined with the donor/acceptor/quencher arrangementsdescribed in the above applications leads to useful FPE quenching andenergy transfer mechanism, which further enhance and expand theusefulness of the techniques for DNA hybridization and other molecularanalysis.

Electronic Perturbation Catalysis

The discovery of the fluorescent perturbation effect has alsocontributed to the further discovery of a way to carry out novelelectronic perturbation catalysis. In particular it lead to discoveringa way to over come what is called the leaving group effect in enzymecatalysis. Investigators trying to create synthetic enzyme-likecatalysts have not been able to overcome this obstacle. (see M. J.Heller, J. A. Walder, and I. M. Klotz, Intramolecular Catalysis ofAcylation and Deacylation in Peptides Containing Cysteine and Histidine,J. American Chemical Society, 99, 2780, 1997).

FIG. 12 shows a diagram of a peptide structure containing an arrangementof nucleophilic groups (cysteine-thiol and histidine-imidazole) designedto carry out electronic perturbation catalysis, ester hydrolysis anddeacylation in particular. Two examples of such cysteine and histidinecontaining peptide structures include: Gly-His-Phe-Cys-Phe-Gly andGly-His-Pro-Cys-Pro-Gly. In the example shown in FIG. 12, a cysteine(thiol) and histidine (imidazole) containing catalytic peptide sequenceis immobilized onto the surface (permeation layer) of a microlocation onan active electronic device (via the terminal alpha amino group). Thesystem is designed to catalyze the cleavage of esters and amide bonds(Step 1). The catalytic peptide/device is exposed to a solutioncontaining the particular substrate of interest (ester, amide, etc.),which hydrolyzes and forms an acyl-thiol intermediate (Steps 1 and 2).In general, the acyl-thiol group will not deacylate even when theimidazole group is in close proximity, because of the back attackbetween the two nucleophiles (Step 3). Electronic perturbation catalysisis carried out by applying an appropriate electronic pulsing sequence(current, on time/off time), which causes charge separation between thenegatively charged thiol group and the positively charged acyl-imidazolegroup (Step 4), allowing the acyl-imidazole group to effectivelydeacylate before the thiol group can re-attack (Step 5). The system isnow ready to catalyze the hydrolysis of a new substrate molecule (Step6). This example represent just one of many possible catalyticarrangements and applications for electronic perturbation catalysis.

Experimental Results

EXAMPLE 1 Ras G Match/Mismatch

APEX Chip Preparation and Capture Probe Loading—APEX active DNA chips,with 25 microlocation test sites (80 microns in diameter) were coatedwith streptavidin agarose accordingly. A 2.5% glyoxal agarose (FMC)solution in water was made according to manufacturer's instructions. Thestock was equilibrated at 65° C., for 5 minutes. Chips were spin coatedat 2.5K rpm for 20 seconds. Another layer was then applied at 10K rpmfor 20 seconds. This second “thin layer was composed of a 1:4 mix of 5mg/ml streptavidin (BM) in 50 mM NaPhosphate, 250 mM NaCl and 2.5%glyoxal agarose.

The chips were baked at 37° C. for 30 minutes. Streptavidin was coupledto the agarose via Schiff's base reduction in 0.1M NaCNBH3 in 0.3MNaBorate, pH 9.0, for 60 minutes, at room temperature. The remainingaldehydes were capped with 0.1M glycine, for 30 minutes, at roomtemperature, and finally rinsed in water, dried under N₂ and then storedat 4° C.

The table below gives the sequence and labeling positions for all theoligonucleotide probes and target sequences used in examples 1 and 2.Mismatches are underlined and bolded. Modi- fied Name Sequence (5′-3′)Modification end Ras 411 GCCCACACCGCCGGCGCCCACC Bodipy 5′ Texas Red Ras415 GGTGGGCGCCGGCGGTGTGGGC Biotin 5′ Ras 416 GGTGGGCGCCGG A GGTGTGGGCBiotin 5′ HLA 253 CCACGTAGAACTGCTCATC Bodipy 5′ Texas Red HLA 241GATGAGCAGTTCTACGTGG Biotin 3′ HLA 378 GATGAGCAG C TCTACGTGG Biotin 3′HLA 375 T ATGAGCAGTTCTACGTGG Biotin 3′ HLA 376 GATGAGCAGTTCTACGTG TBiotin 3′ HLA 401 GATGAGCAGTTCTACGTGG Biotin 5′

Capture Probe Addressing for Example 1—Columns 1 & 2 on the APEX chipwere electronically addressed with the Ras 415 (match) sequence andcolumns 4 & 5 loaded with Ras 416 (mismatch) sequence. Addressing wascarried out in 50 mM cysteine, 1□M oligonucleotide, 200 nA for 1 min.The target/reporter sequence Ras 411 was passively hybridized in 500 mMNaCl, 50 mM NaPhosphate pH 7.4, at room temperature for 5 minutes).Electronic dehybridization and stringency was done at 1.5μA/microlocation, DC pulsing for 0.1 sec on, 0.2 sec off, 150 cycles (20mM NaPhosphate, pH 7.4). Microlocations were given electronic stringencyindividually. Fluorescence signal was captured at 1 second intervals.Normalized displayed is the average of three test sites for each point.Error bars are standard deviations. Results are shown in FIG. 5.

EXAMPLE 2 Ras G and HLA Match/Mismatches

The APEX chip preparation procedure was the same as Example 1. Captureprobe addressing conditions were the same as Example 1. The Ras 415sequence was electronically addressed to all 5 microlocations in column1 and Ras 416 addressed to all 5 microlocations in column 2 of the APEXchip. The HLA 241 sequence was addressed to all 5 microlocations incolumn 4 and HLA 378 was addressed to all 5 microlocations in column 5.The Ras 411 and HLA 253 fluorescent target probes were mixed andpassively hybridized to the APEX chip. Electronic dehybridization andstringency was carried out for the Ras system at 1.5 μA/microlocation,DC pulsing for 0.1 sec on, 0.2 sec off, 150 cycles (20 mM NaPhosphate,pH 7.4). Electronic dehybridization and stringency for the HLA systemwas carried out at 0.6 μA/microlocation, DC pulsing for 0.1 sec on, 0.2sec off, 150 cycles (20 mM NaPhosphate, pH 7.4). Data collected asreported above. FIG. 6 shows the results for Example 2.

EXAMPLE 3 Fluorescent Perturbation Effect with Single Fluorophore

APEX Chip Preparation and Capture Probe Loading—APEX active DNA chips,with 25 microlocation test sites (80 microns in diameter) were coatedwith streptavidin agarose accordingly. A 2.5% glyoxal agarose (FMC)solution in water was made according to manufacturer's instructions. Thestock was equilibrated at 65° C., for 5 minutes. Chips were spin coatedat 2.5K rpm for 20 seconds. Another layer was then applied at 10K rpmfor 20 seconds. This second “thin layer was composed of a 1:4 mix ofmg/ml streptavidin (BM) in 50 mM NaPhosphate, 250 mM NaCl and 2.5%glyoxal agarose. The chips were baked at 37° C. for 30 minutes.Streptavidin was coupled to the agarose via Schiff's base reduction in0.1M NaCNBH₃ in 0.3M NaBorate, pH 9.0, for 60 minutes, at roomtemperature. The remaining aldehydes were capped with 0.1M glycine, for30 minutes, at room temperature, and finally rinsed in water, driedunder N2 and then stored at 4° C.

The sequences for the oligonucleotide reporter probe, quencher probe andcapture probe used in Examples 3 and 4 are listed below: QATAR-1(perfect match for reporter and quencher) 5′-biotin-CAC gAg AgA CTC ATgAgC Agg ggC TAg CCg ATC ggg TCC TCA ggT CAA gTC QATAR-2 5′-biotin-CACgAg AgA CTC ATg AgC Agg (C)gC TAg CCg ATC ggg TCC TCA ggT CAA gTCQATAR-3A (1 base mismatch) 5′-biotin-CAC gAg AgA CTC ATg AgC Agg ggC TAgCC( A ) ATC ggg TCC TCA ggT CAA gTC QATAR-4A (2 base mismatch)5′-biotin-CAC gAg AgA CTC ATg AgC Agg ggC TAg CC(A) A(C)C ggg TCC TCAggT CAA gTC QATAR-5A (perfect match to reporter, no quencherhybridization) 5′-biotin-gCA CCT gAC TCC TgA ggA gAA gTC CCg ATC ggg TCCTCA ggT CAA gTC ET60-BODIPY TR (Reporter) 5′-TgA CCT gAg gAC CCg ATC g -BODIPY TR ET71-Malachite Green (Quencher) 5′-malachite green-Ag CCC CTgCTC ATg AgT CTC T

The capture probes were addressed to specific microlocation test sites(pads) on the APEX chip as follows: a 10 μl aliquot containing 500 nMcapture probe in 50 mM histidine buffer was applied to the chip andpositive bias was applied at 200 nA/pad, for 30 seconds. The bias wasturned off and the chip was fluidically washed in 50 mM histidine.QATAR-1 was addressed to column 1, QATAR-3A was addressed to column 2,QATAR-4A was addressed to column 3, and QATAR-5 was addressed to column4.

Hybridization and Quenching Efficiency

The addressed APEX chips were passively hybridized with ET60-BTRreporter with/without ET71-MG quencher at 500 nM each in 100 mMNaPhosphate, at pH 7.2, 250 mM NaCl, at 65° C. in a heat block, for 2minutes. The chips were washed in 20 mM NaPhosphate, pH 7.2, at roomtemperature, 3 times for 10 minutes each wash. Capture Reporter ET60-BTRQuencher ET71-MG QATAR-1 match match QATAR-3A 1 base pair mismatch matchQATAR-4A 2 base pair mismatch match QATAR-5 match none

Comparison of hybridization signal intensities indicated thatfluorescent quenching was about 50% efficient. This could be improvedwith optimized spacing and or increased purification of the probes(higher specific activity).

Fluorescence Perturbation for Reporter Probe Only

The chips were mounted on a probe station with a probe card to provideelectrical contact to the chip, waveforms were supplied by KeithleyPower Supply, images acquired via Optronics cooled color CCD and NIHimage software was used to analyze the data. The preferred imagingsystem is that disclosed in copending U.S. application entitled“Scanning Optical Detection System”, filed May 1, 1997, incorporatedherein by reference as if fully set forth herein.

Chips were prepared and hybridized as described in Example 1 and 2. In20 mM NaPhosophate, pH 7.2, individual pads were biased negative and apulse waveform was applied. Parameters tested were pulse frequency, %duty cycle, and amplitude. Good fluorescence perturbation results wereobserved at 600 nA/1 sec On/1.5 sec Off. The camera integration was 1.0second. Higher pulse frequencies could also be effective but theseexperiments were limited by the amount of fluorescence at each padlocation which necessitated longer camera integration times.

Results from the perfect match reporter/quencher pair on QATAR 1 showedapprox 10% increase in fluorescence intensity when the power was firstapplied and the intensity oscillated during the course of the waveform.On QATAR-5 which did not have the quencher hybridized there was verylittle fluorescence perturbation. Both QATAR 3a and 4a some fluorescenceperturbation but not as much as QATAR1. Additionally, signal loss afterbias was greatest for QATAR-4A, followed by 3A, followed by 5 andthen 1. This would be expected based on the hybrid Tm's. The results forQATAR-1 (match) and the QATAR-3 (mismatch) are shown in FIGS. 7A and 7B.

EXAMPLE 4 Fluorescence Perturbation with Reporter and Quencher Probes

APEX chips were prepared and hybridized as described in Examples 1, 2,and 3. Microlocation test sites were biased as in Example 3 except thatthe CCD camera integration was 0.5 seconds. Results showed that QATAR-1produced approximately 60% increase in fluorescence intensity when powerfirst applied and intensity oscillated during the entire waveform. ForQATAR-5, which did not have the quencher when hybridized, there was verylittle fluorescence perturbation. Both QATAR 3A and 4A showed an initialincrease in fluorescence approaching 40%. There was a significantdecrease in intensity on QATAR-4A after bias applied. This is indicativeof the lower Tm of this hybrid which had 2 mismatches. The results forQATAR 1 (match) and QATAR 3 (mismatch) are shown in FIG. 9.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it may be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method for catalyzing a cleavage of a bond with an electric fielddevice, comprising the steps of: coupling a catalytic peptide sequenceto an electrode, wherein the catalytic peptide sequence has a firstreactive group and a second reactive group; contacting the catalyticpeptide sequence that is coupled to the electrode with a solutioncontaining a substrate to be hydrolyzed; reacting the first reactivegroup with the substrate to form a first intermediate; reacting thesecond reactive group with the first intermediate to form a positivelycharged second intermediate and a negatively charged first reactivegroup, wherein the positively charged second intermediate comprises anacyl bond; applying an electronic pulsing sequence to the electrode toseparate the negatively charged first reactive group from the positivelycharged second intermediate; and reacting the second intermediate byacyl transfer to cleave the acyl bond.
 2. The method of claim 1, whereinthe first reactive group is a sulfhydryl.
 3. The method of claim 1,wherein the first reactive group is a deprotonated sulfhydryl group. 4.The method of claim 3, wherein the deprotonated sulfhydryl group isformed from a de-protonated cysteine.
 5. The method of claim 1, whereinthe second reactive group is an imidazole.
 6. The method of claim 5,wherein the imidazole is part of a histidine.
 7. The method of claim 1,wherein the acyl bond is an amide.
 8. The method of claim 1, wherein thecatalytic peptide is coupled to a permeation layer associated with theelectrode.
 9. The method of claim 1, wherein the substrate comprises anester.
 10. The method of claim 1, wherein the substrate comprises anamide.
 11. The method of claim 1, wherein the step of reacting thesecond intermediate by acyl transfer to cleave the acyl bond compriseshydrolyzing the acyl bond.
 12. A method for catalyzing cleavage of abond with an electric field device, comprising the steps of: coupling acatalytic peptide sequence to an electrode, wherein the catalyticpeptide sequence has a deprotonoated sulfhydryl group and an imidazole;contacting the catalytic peptide that is coupled to the electrode with asolution containing a substrate to be hydrolyzed; reacting thedeprotonoated sulfhydryl group with the substrate to form a firstintermediate; reacting the imidazole with the first intermediate to forma positively charged second intermediate and a negatively chargeddeprotonoated sulfhydryl group, wherein the positively charged secondintermediate comprises an acyl bond; applying an electronic pulsingsequence to the electrode to separate the negatively chargeddeprotonoated sulfhydryl group from the positively charged secondintermediate; and reacting the second intermediate by acyl transfer tocleave the acyl bond.
 13. The method of claim 12, wherein thedeprotonoated sulfhydryl group is formed from a de-protonated cysteine.14. The method of claim 12, wherein the imidazole is part of ahistidine.
 15. The method of claim 12, wherein the acyl bond is anamide.
 16. The method of claim 12, wherein the catalytic peptide iscoupled to a permeation layer associated with the electrode.
 17. Themethod of claim 12, wherein the step of reacting the second intermediateby acyl transfer to cleave the acyl bond comprises hydrolyzing the acylbond.
 18. The method of claim 12, wherein the substrate comprises anester.
 19. The method of claim 12, wherein the substrate comprises anamide.
 20. A method for catalyzing cleavage of a bond with an electricfield device, comprising the steps of: coupling a catalytic peptidesequence to an electrode, wherein the catalytic peptide sequence has athiol and an imidazole; contacting the catalytic peptide that is coupledto the electrode with a solution containing a substrate to behydrolyzed; reacting the thiol with the substrate to form a firstintermediate; reacting the imidazole with the first intermediate to forma positively charged second intermediate and a negatively chargeddeprotonated thiol, wherein the positively charged second intermediatecomprises an acyl bond; applying an electronic pulsing sequence to theelectrode to separate the negatively charged deprotonated thiol from thepositively charged second intermediate; and reacting the secondintermediate by acyl transfer to cleave the acyl bond.
 21. The method ofclaim 20, wherein the thiol is part of a cysteine.
 22. The method ofclaim 20, wherein the imidazole is part of a histidine.
 23. The methodof claim 20, wherein the acyl bond is an amide.
 24. The method of claim20, wherein the catalytic peptide is coupled to a permeation layerassociated with the electrode.
 25. The method of claim 20, wherein thestep of reacting the second intermediate by acyl transfer to cleave theacyl bond comprises hydrolyzing the acyl bond.
 26. The method of claim20, wherein the substrate comprises an ester.
 27. The method of claim20, wherein the substrate comprises an amide.